Chlorine-Doped Graphene Quantum Dots with Enhanced Anti- and

May 23, 2019 - Here, we report chlorine-doped GQDs (Cl-GQDs) with a tunable Cl doping amount and improved anti- and pro-oxidant activities...
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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 21822−21829

Chlorine-Doped Graphene Quantum Dots with Enhanced Anti- and Pro-Oxidant Properties Lifeng Wang,† Yan Li,*,† Yingmin Wang,† Wenhui Kong,† Qipeng Lu,† Xiaoguang Liu,† Dawei Zhang,† and Liangti Qu‡ †

School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China Beijing Key Laboratory of Photoelectronic/Electrophotonic Conversion Materials, Key Laboratory of Cluster Science, Ministry of Education, School of Chemistry, Beijing Institute of Technology, Beijing 100081, P. R. China

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ABSTRACT: Production or elimination of highly reactive oxygen species is critical in antioxidant, photodynamic, therapeutic, and antibacterial applications. Recent studies have demonstrated that graphene quantum dots (GQDs) possess anti- and pro-oxidant properties simultaneously. However, their efficiency is low. Here, we report chlorine-doped GQDs (Cl-GQDs) with a tunable Cl doping amount and improved anti- and pro-oxidant activities. The scavenging performance and the free radical-produced efficiency of Cl-GQDs are about 7-fold and 3-fold, respectively, higher than those of the undoped GQDs. Meanwhile, Cl-GQDs are considered to be promising for antibacterial applications because of their enhanced singlet oxygen generating ability. We hope that this study could provide a new strategy to develop nanomaterials for application in the anti- and pro-oxidant field. KEYWORDS: graphene quantum dots, heteroatom doping, enhanced antioxidant, antibacterial

1. INTRODUCTION Reactive oxygen species (ROS), including singlet oxygen (1O2), superoxide anion (O2•−), and hydroxyl radicals (•OH), are derived from molecular oxygen containing one or more unpaired electrons.1 The unpaired electrons endow them with high reactivity. A suitable ROS content is essential for normal cell in physiological processes, such as proliferation and homeostasis.2 However, a burst of ROS in cells could cause oxidative damage to cell membranes, protein structures, and DNA, and probably induce cancer-causing mutations.3−7 Therefore, antioxidant materials are essential to maintain the balance of the ROS content. From another perspective, high levels of ROS generated from photosensitizers by light excitation could kill cancer cells because they are more vulnerable to exogenous ROS than ordinary cells.1,8 And ROS could also cause membrane damage of bacterial cells to exhibit antimicrobial activity.9,10 Accordingly, highly efficient ROSgenerating materials will also be required for photodynamic therapy and antibacterial fields. Several groups reported that carbon nanomaterials [including fullerenes, carbon nanotubes (CNTs), graphene, and functionalized carbon dots] could scavenge ROS by hydrogen donation, adduct formation, or electron transfer. 11−15 Furthermore, carbon nanomaterials with defects or heteroatom sites could also generate ROS through surface reactions under light irradiation, which often involves defect or heteroatom sites.16,17 Graphene quantum dots (GQDs), small fragments of graphene sheet containing abundant surface functional groups, © 2019 American Chemical Society

have attracted more interest owing to their outstanding optical properties and favorable biocompatibility.18,19 Especially, the unpaired electrons from the defects and the π-conjugated nature of GQDs facilitate their electron storage capacity and charge transfer abilities, which endow them with free radical scavenging properties.4,20 Meanwhile, under a blue laser or white light irradiation, GQDs could generate 1O2, exhibiting pro-oxidant properties.10,21 Compared with cytotoxic CNTs or graphene oxide, small sized GQDs, featuring superior biocompatibility and photoluminescence (PL) property, have become one of most promising and marketable antioxidants or pro-oxidants in organisms. However, the anti- and pro-oxidant activities of GQDs have been far from meeting the requirements of application until now because the surface functional groups and lattice defects limit the unmodified GQDs. Also, only parts of the defects are active for producing or scavenging free radicals.21 Heteroatom doping is an effective method to modify the defect level of GQDs and regulate their physical and chemical properties. The heteroatom dopants could create positive or negative charges on adjacent carbon atoms in the graphitic lattice. In addition, the corresponding electronic density and charge transfer ability of GQDs could be varied, which lead to the increase of fluorescence in the intensity or catalytic Received: February 22, 2019 Accepted: May 23, 2019 Published: May 23, 2019 21822

DOI: 10.1021/acsami.9b03194 ACS Appl. Mater. Interfaces 2019, 11, 21822−21829

Research Article

ACS Applied Materials & Interfaces

200 μL of the sample solutions containing 20 μL TEMP and exposure to a 450 W xenon lamp (1000 W/m2) for 10 min. The control group was conducted in almost the same strategy except the light irradiation. Cyclic voltammetry (CV) curves were carried out on a CHI 660D electrochemical working station (Chenhua Instrument, Shanghai, China) with a three-electrode system. Cl-GQDs modified glassy carbon electrode (GCE) was used as the working electrode. Pt plate and Ag/AgCl electrodes were used as the counter and reference electrodes, respectively. GCE was polished with alumina powders and ultrasonication in ethanol and deionized water before the experiment. 50 μL Cl-GQDs (200 μg/mL) solutions were spread on GCE and dried naturally in air. All the electrochemical measurements were recorded in 0.1 M KCl solution containing 0.5 mM K3[Fe(CN)6] and 0.5 mM K4Fe(CN)6. 2.3. Radical Scavenging Assays and Antioxidant Activity. DPPH radical (DPPH•) scavenging assay. 1,1-Diphenyl-2-picrylhydrazyl radical (Shanghai Jinsui Biotechnology Co., Ltd., China, 97% in purity), as a stable nitrogen-centered free radical, was used to evaluate the free radicals scavenging performance of Cl-GQDs. 1 mL ethanol solution containing 100 μM DPPH• was mixed with 1 mL Cl-GQDs (0−300 μg/mL) aqueous solution after being incubated in the dark for 1 h. The scavenging efficiency was measured according to the decrease of absorption intensity at 520 nm. KMnO4 reduction assay. KMnO4 (Sinopharm Chemical Reagent Co., Ltd., China, 99.5% in purity) could also evaluate the antioxidant activity of GQDs following the protocol by Ruiz.5 2 mL acidified (pH = 3) KMnO4 solutions (100 mM) containing 100 μg Cl-GQDs were incubated in the dark for 30 min. After reduction by antioxidants, the KMnO4 solutions changed from purple to colorless. The remaining concentration of KMnO4 was monitored by UV−vis absorption spectroscopy at 515 nm. Hydroxy radical scavenging assay. •OH could be produced by a photochemical reaction of TiO2 nanoparticles (P25, Degussa AG Co., Ltd.). Terephthalic acid could capture •OH and transform it into 2hydroxy terephthalic acid (λex: 315 nm and λem: 430 nm), which could be used as a fluorescence probe to determine the concentration of • OH. Therefore, in this paper, 2 mL solutions containing 25 mM phosphate-buffered saline (PBS), 0.5 mM terephthalic acid, 100 μg TiO2, and 100 μg Cl-GQDs were irradiated by UV light (8 W, 365 nm) for 1 h. Finally, the concentration of •OH could be monitored by the intensity of the PL spectrum (430 nm). Dye protect assay. Rhodamine B (RhB, Sinopharm Chemical Reagent Co., Ltd., China) was used as a model target molecule for an oxidant attack. 2 mL solutions containing 25 mM PBS, 50 μg/mL TiO2 (P25), 10 μM RhB, and 100 μg/mL Cl-GQDs were irradiated by UV light (8 W, 365 nm) for 3 h with magnetic stirring. The concentration of the remaining RhB was monitored by its absorption spectrum (552 nm). 2.4. Pro-Oxidant Assay. Ascorbic acid (AA) oxidation assay. Prooxidant assay was conducted according to the protocol by Chong,4 in which AA (Xilong Scientific Co., Ltd., China, 99.7% in purity) could be oxidized by the photoexcited GQDs under an excitation light of a 450 W xenon lamp (1000 W/m2). Briefly, 1 mL AA solution (100 μM) was mixed with 1 mL Cl-GQDs (100 μg/mL) aqueous solution under light irradiation for 30 min. Then, the remaining AA could be monitored by the absorption intensity (λmax = 265 nm). The prooxidant performance of Cl-GQDs was evaluate by the peak intensity of AA. Antibacterial assay. Escherichia coli (ATCC 25922) was cultured overnight in a Luria-Bertani (LB) agar medium at 37 °C to the stationary phase. Then, the bacterial cells were centrifuged at 3000 rpm for 5 min, washed twice, and subsequently diluted with 0.9% sterile saline (OD600 = 0.7). After that, 200 μL bacterial suspension was diluted to 5 mL sterile saline, in which Cl-GQDs-7.5V were added until their concentration reaches 50 μg/mL. The control experiment was conducted under almost the same conditions except that no Cl-GQDs-7.5V was added. The two as-prepared bacterial suspensions were exposed to the simulated sunlight (1000 W/m2) for 2 h, centrifuged at 3000 rpm for 5 min, and subsequently redispersed in the sterile saline. Bacterial suspensions containing Cl-GQDs-7.5V

activity.22,23 Furthermore, the polarized carbon atoms could enhance the antioxidant performance.24 Besides, related research also pointed out that the ROS-generating ability of GQDs was closely related to the ketonic carbonyl groups.25 It seems that if a high level of defects can be introduced into the GQDs by doping and the oxygen-containing functional groups can be optimized to generate carbonyl groups, enhanced antiand pro-oxidant properties could be expected. However, related research has been rarely reported. Here, we realized the regulation of the surface functional group (i.e. CO/COOH group) in the synthesis of chlorinedoped GQDs (Cl-GQDs). Cl was chosen as the dopant because of its relatively large atom radium and high electronegativity compared to the carbon atom, which favors the electronic interaction between Cl-GQDs and free radicals. Although Cl-GQDs have been prepared and their fluorescence and electron transfer properties in the photovoltaic detector have been investigated elsewhere,26−28 their preparation processes are normally energy intensive, highly risky, and thus hardly commercially available (∼450$/100 mg). Therefore, in this work, an electrochemical approach was proposed to synthesize Cl-GQDs and a widely used artificial sweetener (i.e. sucralose) was employed to provide the C and Cl source. This method is not only facile and economic, but can also realize the regulation of surface functional groups (i.e. CO/ COOH) and Cl-doping concentration during the preparation simultaneously. Through optimizing the amount of Cl dopant and surface CO/COOH, the as-prepared Cl-GQDs exhibited higher activity for different free radicals scavenging than the undoped ones. Meanwhile, they could also produce ROS more efficiently under light irradiation. As an antibacterial agent, the ratio of antibiosis thereof could reach to 96.6%. We hope our work can be helpful for the controlled synthesis of Cl-GQDs and designing of anti- and pro-oxidant materials with high activity.

2. EXPERIMENTAL SECTION 2.1. Preparation of Cl-GQDs. Cl-GQDs were prepared by an electrochemical method using a CHI 660D working station. Two platinum wires were used as the working and countering electrode, respectively. 1 g sucralose (Shanghai Yuanye Bio-Technology Co., Ltd., China, 98% in purity) was dissolved in 10 mL NaOH (4 M) aqueous solution, which provided the C and Cl sources. The amount of oxygen-containing functional groups and Cl dopants were adjusted by changing the voltage and electrolysis time in the synthesis procedure. Cl-GQDs-5V and Cl-GQDs-7.5V were obtained by a constant potential method with 5 and 7.5 V voltage supply for 45 min. Cl-GQDs-5V-2h was obtained by a similar method with 5 V voltage supply for 2 h. The Cl-GQDs were collected by filtering the electrolyte with a cellulose filtration membrane (0.22 μm) and dialyzing with a cellulose ester membrane bag (retained molecular weight: 3500 Da) for 6 days. Besides, the compared GQDs were prepared by the constant-voltage scanning procedure as reported in our previous work.21 2.2. Material Characterization. Transmission electron microscopy (TEM) observation was carried out using an H-7650B electron microscope at 120 kV. UV−vis absorption and PL spectra were measured using a TU-1900 spectrophotometer and a fluorimeter (Hitachi F-7000), respectively. X-ray photoelectron spectroscopy (XPS) was performed on an ESCALAB 250Xi electron spectrometer with an Al monochromatic Kα radiation (hν = 1486.6 eV) source. The energies of all the spectra were calibrated with respect to the C 1s peak at 284.5 eV. Electron-spin resonance (ESR) measurements were carried out by using an ESR spectrometer (A300, Bruker). The spin trap 2,2,6,6tetramethylpiperidine (TEMP) was selected to detect singlet oxygen. 21823

DOI: 10.1021/acsami.9b03194 ACS Appl. Mater. Interfaces 2019, 11, 21822−21829

Research Article

ACS Applied Materials & Interfaces

Figure 1. (a) Schematic illustration of the preparation of Cl-GQDs. TEM images of Cl-GQDs-5V (b), Cl-GQDs-7.5V (c), and Cl-GQDs-5V-2h (d). (e) HRTEM image of Cl-GQDs-5V.

Figure 2. (a) PL spectra of the Cl-GQDs-5V, Cl-GQDs-7.5V, Cl-GQDs-5V-2h, and GQDs dispersed in water at room temperature. The inserts show the photograph (left) and fluorescence images of the Cl-GQDs-7.5V solutions under UV light of 365 nm (right). FT-IR spectra (b) and XPS survey spectra (c) of the Cl-GQDs-5V, Cl-GQDs-7.5V, Cl-GQDs-5V-2h, and GQDs. (d) Cl 2p high-resolution XPS spectrum of Cl-GQDs-7.5V. in the dark was conducted in the same strategy. After that, 100 μL of 100-fold serial dilutions bacterial suspensions were coated on a LB agar plate, respectively, and incubated at 37 °C overnight to observe the formation of colonies.

The UV−vis spectrum of Cl-GQDs, shown in Figure S1, displays a typical absorption band at ca. 268 nm and a small absorption tail that extended to 350 nm attributing to the π−π* transition of the CC and the n−π* transition of oxygen-containing groups, respectively. The light-yellow ClGQDs aqueous solution emitted a strong blue-green fluorescence under irradiation at 365 nm (Figure 2a, inset). With heteroatoms doping, Cl-GQDs exhibited s stronger PL intensity compared with GQDs in the same concentration, which could be ascribed to the interplay between the dopants and the carbon atoms in the graphene lattice.23,29 The Fourier transform infrared (FT-IR) spectra of the Cl-GQDs (Figure 2b) shows peaks at 588, 710, and 788 cm−1 owing to the C−Cl stretching vibration, which confirms the successful incorporation of chlorine atoms into GQDs. The other peaks at 1048, 1720, and 3430 cm−1 are ascribed to C−O−C, CO, and −OH stretching vibration, respectively. Furthermore, the

3. RESULTS AND DISCUSSION 3.1. GQDs Characterization. Cl-GQDs were prepared through the electrochemical treatment of sucralose, as illustrated in Figure 1a. TEM and high-resolution TEM (HRTEM) measurements were performed to investigate the morphology of Cl-GQDs. Figure 1b−d show that the asprepared Cl-GQDs could be uniformly dispersed, which was similar to the undoped GQDs previously reported.18 From the HRTEM result of Cl-GQDs in Figure 1e, a clear lattice fringe of 0.24 nm matches with the (1120) facet of graphite. This suggests that the doped chlorine atoms do not influence the graphite nature and the surface morphology of Cl-GQDs. 21824

DOI: 10.1021/acsami.9b03194 ACS Appl. Mater. Interfaces 2019, 11, 21822−21829

Research Article

ACS Applied Materials & Interfaces

Figure 3. DPPH• scavenging assay. (a) DPPH• remaining ratio after being incubated with Cl-GQDs-5V, Cl-GQDs-7.5V, Cl-GQDs-5V-2h, and GQDs in the dark for 30 min. (b) Absorption spectra of 2 mL DPPH• solutions (50 μM) containing 0−300 μg Cl-GQDs-7.5V after incubation in the dark for 1 h. Error bars represent the standard deviation.

Figure 4. KMnO4 reduction and hydroxyl radical scavenging assays. (a) KMnO4 remaining ratio after incubated with Cl-GQDs-5V, Cl-GQDs-7.5V, Cl-GQDs-5V-2h, and GQDs in the dark for 1 h (pH = 3). (b) Hydroxyl radical remaining ratio after incubated with Cl-GQDs-5V, Cl-GQDs-7.5V, Cl-GQDs-5V-2h, and GQDs under light irradiation for 1 h.

nanomaterials from the literature, such as GO and pC60 or modified CNT,6,30 Cl-GQDs-7.5V exhibit a higher reaction rate and a superior antioxidant activity. The DPPH • scavenging mechanism is discussed in detail later. 3.2.2. KMnO4 Reduction and Hydroxyl Radical Scavenging Assay. KMnO4 reduction assay was further conducted to evaluate the antioxidant activity of Cl-GQDs. Mn7+ in acidified KMnO4 solutions could be reduced to Mn2+ after antioxidants were added, which is accompanied by a decreased absorbance at 515 nm.31 The antioxidant property of each sample could be estimated compared with the characteristic absorption peak of KMnO4 solutions without any antioxidants. Figure 4a shows the KMnO4 remaining ratio with different antioxidants after 1 h reaction in the dark. It could be noticed that KMnO4 was slightly reduced by GQDs according to the absorption spectra, but KMnO4 solutions with all Cl-GQDs exhibited more significant reduction, which indicated that Cl doping played an important role in the KMnO4 reduction assay. On the other hand, similar to the DPPH• assay, the order of the reducing capacity is: Cl-GQDs-7.5V > Cl-GQDs-5V > Cl-GQDs-5V-2h. These results further confirmed that the content of Cl atoms in Cl-GQDs is also very important to their antioxidant property. Our previous studies confirmed that Fe2+ could be chelated with GQDs and thus depressed the Fenton reaction.32 Furthermore, we confirmed that there was no •OH generated from Cl-GQDs through a PL test by using terephthalic acid as a fluorescence probe under UV light irradiation (365 nm). Hence, we employed a photochemical reaction of TiO2 nanoparticles to produce •OH. Terephthalic acid could capture the •OH and transform it into a fluorescence 2-hydroxy terephthalic acid. The remaining ratio of •OH was evaluated by the PL intensity. As illustrated in Figure 4b, the PL intensity of terephthalic acid solution containing TiO2 nanoparticles and GQDs decreased to various degrees after UV light irradiation

Raman spectra (in Figure S2) revealed that a large number of defects were produced in the Cl-GQDs, given by the Cl doping. XPS measurements further confirm the successful doping of Cl atoms in Cl-GQDs-5V, Cl-GQDs-7.5V, and Cl-GQDs-5V2h from Figure 2c, in which a pronounced Cl 2p peak at ca. 199 eV is observed along with the C 1s (ca. 284 eV) and O 1s (ca. 530 eV) main peaks. During the synthesis process, we prepared Cl-GQDs with tunable amounts of Cl by changing the voltage or electrolysis time. Cl contents in Cl-GQDs-5V, Cl-GQDs-7.5V, and Cl-GQDs-5V-2h are 0.89, 1.32, and 0.62%, respectively (Table S1). In addition, the high-resolution C 1s spectrum of each sample (Figure S3) indicates that the CO/COOH content rises with the increase of voltage or the duration of the electrolysis. 3.2. Antioxidant Activity. 3.2.1. DPPH Radical Scavenging Assay. The antioxidant activity of Cl-GQDs was first investigated by using DPPH• as the model radical. As a relatively stable free radical, DPPH• has been commonly used to assess the antioxidant activity of compounds. A stable DPPH−H complex could be formed when an antioxidant was added and the color immediately changed from deep purple to pale yellow/colorless. Figure 3a shows the DPPH• remaining ratio of each GQDs after incubation in the dark for 30 min. Comparing with undoped GQDs, all of the Cl-GQDs samples show higher antioxidant activity. Meanwhile, the antioxidant activity is dependent on the Cl doping content, From ClGQDs-5V-2h (Cl content is 0.62%), Cl-GQDs-5V (Cl content is 0.89%) to Cl-GQDs-7.5V (Cl content is 1.35%), the antioxidant activity was gradually improved. In addition, from the variation of the intensity of the characteristic peaks in DPPH• absorption spectra, as shown in Figure 3b, the antioxidant activity of Cl-GQDs-7.5V is also related to their concentration. Furthermore, compared with other carbon 21825

DOI: 10.1021/acsami.9b03194 ACS Appl. Mater. Interfaces 2019, 11, 21822−21829

Research Article

ACS Applied Materials & Interfaces

Figure 5. Dye protect assay. (a) RhB (10 μM) remaining ratio after photocatalytic degradation with or without antioxidant protection. (b) Photo of the solutions after the photocatalytic experiment.

Figure 6. (a) AA remaining ratio after light irradiation for 30 min. (b) O 1s high-resolution XPS spectra of Cl-GQDs-5V, Cl-GQDs-7.5V, and ClGQDs-5V-2h. (c) Cell viability measurements of E. coli treated with Cl-GQDs. (d) Photographs of LB agar plates contain E. coli bacterial cells with Cl-GQD-7.5V in the dark or exposure to light. Bacterial suspension without Cl-GQD-7.5V was used as the control.

nm). Figure 6a indicates that the ROS generation of the ClGQDs is 3 times more efficient than that of the GQDs. This could be also ascribed to the large number of defective sites in the doped Cl atoms and the corresponding promoted electronic interaction with oxygen. In addition, ROS generation ability is involved with the functional groups such as carbonyl and carboxyl species.25 Carbonyl bonds is an active site to produce ROS, whose content in Cl-GQDs-7.5V is higher than that in Cl-GQDs-5V-2h and Cl-GQDs-5V (Figure 6b). Therefore, the high Cl and carbonyl bond content endow Cl-GQDs-7.5V with the strongest ROS generating ability. Furthermore, ROS are harmful to lipids and proteins of bacteria.33−35 Several studies reported Ag or adenine modified GQDs could be used to kill bacteria, but these GQD-based materials still suffered from inefficient antibacterial performance and complex preparation processes.36,37 Ristic et al. reported that GQDs could kill bacteria under a blue light laser irradiation, but using a laser to kill bacteria is impractical in industrial applications and there were still bacteria that survived at the end.9 In our case, we assessed the antibacterial performance of the Cl-GQDs against E. coli bacteria. First, E. coli suspension with Cl-GQDs-7.5V (50 μg/mL) was exposed to a simulated sunlight for 2 h. In addition, the bacterial cells were coated on a LB agar plate and counted after overnight incubation. Figure 6c,d indicate that the Cl-GQDs-7.5V could

(365 nm). Specifically, the PL intensity of solution with GQDs have decreased slightly (58%), whereas Cl-doped GQDs have decreased dramatically (83, 91, and 78% for Cl-GQDs-5V-2h, Cl-GQDs-5V, and Cl-GQDs-7.5V, respectively), indicating the Cl-GQDs with more Cl possess higher •OH scavenging efficiencies. 3.2.3. Dye Protection Assay. Furthermore, Cl-GQDs were selected to investigate their potential to prevent the degradation of organic dyes from oxidation pressure. In our case, TiO2 nanoparticles were selected again to produce ROS by a photochemical reaction. Once antioxidants were added, the degradation of RhB by ROS was suppressed. In addition, the antioxidant property of Cl-GQDs could be evaluated by the remaining ratio of RhB, which was detected by the absorption spectra. As shown in Figure 5a, the remaining ratio of RhB (84.4%) is the highest in the solution containing ClGQDs-7.5V among all samples. The dye-protection efficiency of Cl-GQDs-7.5V is 7 times higher than that of the undoped one. 3.3. Pro-Oxidant Activity. It is reported that ROS could be produced through energy transfer between GQDs and oxygen molecules.8,33 Also, AA was used as an antioxidant to evaluate the ROS production efficiency of Cl-GQDs. The prooxidant activity of Cl-GQDs was detected by the loss ratio of AA from the change of characteristic absorption peak (265 21826

DOI: 10.1021/acsami.9b03194 ACS Appl. Mater. Interfaces 2019, 11, 21822−21829

Research Article

ACS Applied Materials & Interfaces

Figure 7. (a) C 1s spectra of the Cl-GQDs-5V before and after the reaction with DPPH•. (b) CV curves of the Cl-GQDs samples modified on GCE electrodes in 0.1 M KCl solutions containing 0.5 mM K3[Fe(CN)6] and 0.5 mM K4Fe(CN)6. (c) ESR spectra obtained from the samples containing Cl-GQDs-7.5V and TEMP with (black) or without (red) light irradiation. (d) Schematic illustration of the 1O2 generation mechanism.

To evaluate the influence of Cl in GQDs, CV measurements were conducted by using a 0.1 M KCl solution containing 0.5 mM K3[Fe(CN)6] and 0.5 mM K4Fe(CN)6 as the electrolyte. Cl-GQDs act as an insulating layer on the GCE electrode and hinder the electron transfer between ferricyanide ions and GCE.49 Figure 7b showed a typical redox reaction at the electrode surface with the Cl-GQDs-7.5V exhibiting the highest peak current value than others. Besides, the peak current value decreases with the decreasing Cl doping level, which further indicated that the charge transfer ability of GQDs was closely related to the doped Cl. Hence, the highvalue electronegativity of Cl promoted the electronic interaction between Cl-GQDs and DPPH• and Cl-GQDs7.5V with the highest Cl content possessing the highest DPPH• scavenging efficiency. Similar to DPPH• scavenging, the experimental results of KMnO4 and •OH scavenging assays both demonstrated that the doped Cl atoms promoted the charge transfer between ClGQDs and Mn7+/•OH. Also, the Cl-GQDs with the maximum Cl content exhibiting the strongest reduction property and superior antioxidant ability. The ROS generation from GQDs under light irradiation was confirmed by ESR spectroscopy. TEMP was selected as a spin trap for singlet oxygen, which could selectively react with 1O2.8 After 1O2 was trapped, a stable product 2,2,6,6-tetramethylpiperidine-1-oxyl was produced, leading to a characteristic ESR signal. Figure 7c illustrated a strong ESR signal of TEMP with Cl-GQDs-7.5V solution under irradiation. In addition, no ESR signal was observed for the control sample in the dark. These results demonstrated that 1O2 could be generated by ClGQDs-7.5V under irradiation which agreed well with previous reports.8 Following photoexcitation, 1O2 was produced by energy transfer from the excited triplet state of the Cl-GQDs to the ground-state oxygen (Figure 7d). Also, no O2•− or •OH was generated by GQDs under irradiation.8,33 Compared with GQDs, the higher level of defects induced from the highly

result in a high antibacterial ratio (>96.6%) under a simulated sunlight illumination, while the antibacterial ratio was only 15.3% in the dark conditions. The obtained results indicate the Cl-GQDs-7.5V possesses a high antibacterial ability under a simulated sunlight irradiation. 3.4. Discussion. It is known that carbon atoms in the graphene plane of GQDs are covalently bonded through three electrons with each other and form a strong lattice, but the fourth valence electron on each carbon atom is delocalized. In addition, this delocalized electron could be localized at the edge or defect sites of the GQDs.9 As mentioned before, a lot of sp3 C exist in Cl-GQDs, which leads to the unbalanced distribution of the density of electrons inside the hexagonal carbon ring.38 Moreover, there is a significant difference between electronegativity of chlorine dopant (3.16)39 and carbon atom (2.55).40 Also, the covalently doped electron donating chlorine atoms create net negative charges on adjacent carbon atoms in the graphitic lattice, which further modulate the electronic density and enhance the chemical activity of Cl-GQDs, facilitating the electronic interaction between Cl-GQDs and radicals.41−44 Similar to many heteroatom-doped carbon-based materials (such as CNT, graphene, and GQDs),45−48 Cl-GQDs possess an excellent electron transfer property, which is important for the superior antioxidant and pro-oxidant abilities. The DPPH• scavenging mechanism was further elucidated by the XPS analysis on Cl-GQDs-5V after the reaction with DPPH•. First, the appearance of N in the XPS survey spectra (Figure S5) after the reaction demonstrated that the formation of the adduct is an important mechanism for DPPH • scavenging. Moreover, the C 1s spectra demonstrate that most of the surface C−O bonds are reduced, especially the C− Cl bonds almost disappeared in Cl-GQDs-5V after the scavenging experiment (Figure 7a). This indicated both Cl and O are the reactive center of Cl-GQDs. 21827

DOI: 10.1021/acsami.9b03194 ACS Appl. Mater. Interfaces 2019, 11, 21822−21829

Research Article

ACS Applied Materials & Interfaces

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electronegative Cl atoms facilitated the energy transfer between Cl-GQDs and O2 and thus enhance their pro-oxidant property.

4. CONCLUSIONS In summary, we successfully synthesized Cl-GQDs by a facile electrochemical method. In addition, the Cl-GQDs exhibited an efficiently free radical scavenging ability, which could be ascribed to the high content of defect sites induced by doping with Cl atoms. These Cl-GQDs could scavenge the strong oxidizing ROS and protect dye from degradation. The scavenging performance of Cl-GQDs was about 7 times higher than that of the undoped one. Furthermore, ROS could also be produced by Cl-GQDs under visible light irradiation. The ROS producing capability was about 3 times higher than that of the undoped one. The highly ROS producing ability could also be attributed to the defect induced by Cl doping and their highly CO bond content, which endowed them with an enhanced antibacterial performance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b03194.

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UV−vis absorption spectra, Raman spectra, C 1s highresolution XPS spectra, loss ratio of AA in the dark, and XPS survey spectrum of Cl-GQDs-5V after reaction with DPPH (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yan Li: 0000-0001-5017-4592 Dawei Zhang: 0000-0002-6546-6181 Liangti Qu: 0000-0002-7320-2071 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (grant no. 21674011), Beijing Municipal Natural Science Foundation (2172040), and Fundamental Research Funds for the Central Universities (FRF-GF-17B11).

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DOI: 10.1021/acsami.9b03194 ACS Appl. Mater. Interfaces 2019, 11, 21822−21829

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DOI: 10.1021/acsami.9b03194 ACS Appl. Mater. Interfaces 2019, 11, 21822−21829